Dimethyl sulfoxide

Abstract

The essential oil obtained by hydrodistillation of the leaves of Amomum cannicarpum (Wight) Bentham ex Baker, (Family: Zingiberaceae) was analyzed by GC/FID and GC/MS. Individual components of the oil were identified by their mass spectra which were compared with published work. Twenty-nine compounds were identified, of which the major components were β-pinene (9.0%), caryophyllene oxide (6.6%), β-bisabolene (6.4%) and δ-cadinene (6.2%). The oil showed significant antimicrobial activity against certain Gram-positive and Gram-negative bacteria as well as against two fungi (Candida, albicans and Aspergillus fumigatus).

Key Word Index

Amomum cannicarpum, Zingiberaceae, essential oil composition, antimicrobial activity.

Introduction

Amomum cannicarpum (Wight) Bentham ex Baker (Zingiberaceae) is a stout, gregarious herb growing up to 3.5 m, endemic to south India, fairly common in evergreen forests (1). Amomum cannicarpum leaves were collected from Ponmudi hills, Western Ghats, Kerala, India in July 2001, identified by Mathew Dan, and a voucher specimen No. 36270 was deposited in the Herbarium of the Tropical Botanic Garden and Research Institute (TBGRI).

Although there are reports on the chemical composition of the essential oils from related species such as A. subulatum (2), A. xanthioides (3), A. tsao-ko (3), A. schmidtii (4), A. villosum (5), A. linguiforme (6-7) and A. testaceum (8), there is no report on the chemical composition of the leaf oil of A. cannicarpum. This is the first report on the chemical composition and antimicrobial activity of the leaf oil of A. cannicarpum.

Experimental

Oil isolation and analysis: Fresh leaves (400 g) were hydrodistilled for 3 h using a Clevenger-type apparatus to obtain a sharp, pleasant smelling, pale yellow oil in 0.03% yield. GC/FID analysis of the oil was carried out on a Nucon 5765 gas chromatograph fitted with a SE-30 Chromosorb-W packed stainless steel column (2 m x 2 mm). Oven program: 80°-150°C (8°C/min), 150°-290°C (6°C/min), 290°C (10 min); carrier gas, nitrogen; flow rate 40 mL/min; injector temperature 220°C; detector temperature 240°C. GC/MS analysis of the oil was performed on a Shimadzu GC/MS QP 5050 under the following conditions: splitless injection of 1.0 µL of oil, PB X5 capillary column (30 m x 0.25 mm, 0.25 µm film thickness); carrier gas, helium; flow rate 1 mL/min; injector temperature 250°C; oven temperature 80°-280°C (5°C/min); interface temperature 270°C; mass spectra, electron impact (EI^sup +^) mode 70 eV; and ion source temperature 250°C. Individual components were identified by comparison of mass spectra with those in NIST and Wiley data system libraries and by comparison of retention times with published data. Relative percentage of components was calculated from the peak area percent report of volatiles from GC/FID data (Table I).

Antimicrobial analysis: The leaf oil of A. cannicarpum was tested for its antibacterial and antifungal activities by the disc agar diffusion method (9-10). The following microorganisms were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India as Microbial Type Culture Collection (MTCC) and were used for testing: Gram-positive bacteria Bacillus subtilis (MTCC 441), two different strains of Staphylococcus aureus subsp. aureus (MTCC 704 and 2940), Arthrobacterprotophormiae (MTCC 2682) and Gram-negative bacteria, Serratia marcescens (MTCC 97), Pseudomonas fluorescens (MTCC 103), Escherichia coli (MTCC 443), Salmonella typhi (MTCC 733) and Pseudomonas aeruginosa (MTCC 741). These bacteria were grown on Mueller-Hinton agar medium (pH 7.2 to 7.4). The fungi Candida albicans (MTCC 227) and Aspergillus fumigatus (MTCC 3002) were cultured on modified Sabouraud's agar. Microbial suspensions were then made from the agar plates using relevant broths.

The respective agar media were poured into the plates to uniform depth of 5 mm and allowed to solidify. Then the microbial suspensions were streaked over the surface of media using a sterile cotton swab to ensure the confluent growth of the organism. The disc size used was 6 mm (Whatman No. 1) papers. Aliquots of the oil (10 µL) were diluted with two volumes of dimethyl sulfoxide and impregnated on filter paper discs, which were then aseptically applied to the surface of the agar plates at well spaced intervals. The plates were incubated at 36°C for 24 h and observed zones of inhibition were measured. A control disc impregnated with 10 µL of DMSO (inert solvent), streptomycin and flucanozole (2 µg/disc, references for bacteria and fungi, respectively) were used alongside the test discs in each experiment (Table II).

Results and Discussion

The oil yield from the leaves was 0.03% (v/w). The chemical composition of the leaf oil of A. cannicarpum is presented in Table I. Twenty-nine compounds comprising 88.7% of the leaf oil have been identified. The major chemical constituents were β-pinene (9.0%), caryophyllene oxide (6.6%), β-bisabolene (6.4%) and δ-cadinene (6.2%). The results of the antimicrobial study on the oil are presented in Table II. The oil was found to be moderately active against most of the tested Gram-positive and Gram-negative bacteria and also active against the two tested fungi.

Acknowledgments

The authors express sincere thanks to the director, TBGRI for providing laboratory facilities; C. Arumughan, head, Agroprocessing Division, Regional Research Laboratory, Thiruvananthapuram, India for GC/MS; and University Grants Commission, New Delhi, India for providing fellowship to Joseph Mathew under the Faculty Improvement Program.

References

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8. M. Sirat, L.F. Hong and S.H. Khaw, Chemical composition of the essential oil of the fruits of Amomum testaceum Ridl. J. Essent. Oil Res., 13, 86-87 (2001).

9. G. Cappuccino and N. Sherman, Microbiology: A Laboratory Manuel. pp. 254, Benjamin/Cumming Science Publishing, California (1999).

10. D.A.V. Bershe and A.J. Vlietnick, Screening Methods for Antibacterial and Antiviral Agents from Higher Plants, In: Methods in Plant Biochemistry. Vol. VI, pp 47-69, Edits., P.M. Dey and J.B. Harborne, Academic Press, London (1991).

Joseph Mathew, Baby Sabulal and Varughese George*

Phytochemistry Division, Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram - 695 562, Kerala, India

Mathew Dan

Horticulture and Garden Development Division, Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram - 695 562, Kerala, India

Sugathan Shiburaj

Microbiology Division, Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram - 695 562, Kerala, India

* Address for correspondence

Received: August 2003

Revised: March 2004

Accepted: May 2004

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